1. Field of the Invention
The present invention is in the field of electrochemical detection of organic compounds and more specifically concerns the electrochemical detection of nucleotides and nucleic acid polymers.
2. Background and Summary of the Invention
In the parent to the present application, which is incorporated herein by reference, the use of sinusoidal voltammetry for rapid detection of electroactive neurotransmitters was disclosed. The technique described using either lock-in amplifiers or fast Fourier transformation to detect these substances electrochemically employing miniature electrodes and extremely small sample volumes. These methods have now been extended to permit the detection of nucleic acids such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA).
The sensitive measurement of DNA and RNA is of primary importance due to the preeminent biological significance of these polymers as the primary genetic material and the primary transcriptional information carrier, respectively, of most living organisms. The knowledge of the structure of DNA, and its interactions with other biological compounds like proteins and other small molecular weight compounds might lead to advances in pharmacology, and also to the prevention of many diseases like cancer, sickle-cell anemia and cystic fibrosis. Traditionally, nucleic acids have been detected spectrophotometrically either directly (through UV absorbance of the purine and pyrimidine nucleotide bases) or indirectly through the use of various nucleic acid derivatives. Typical derivatives have included noncovalent labels such as intercalating dyes that operate through insertion into the nucleic acid helix or covalent labels that are directly attached to the nucleic acids which have been chemically derivatized. The added label may permit direct optical detection (i.e., the label is absorptive) or nonoptical detection (i.e., the label is radioactive). The added label may also permit binding of a secondary label such as an antibody which may itself be optically or radioactively labeled. It will be appreciated that derivatization-based methods are slow, complex and may result in sample loss or damage. On the other hand, direct optical detection is usually of insufficient sensitivity.
There is considerable need for extremely rapid, sensitive methods for nucleic acid detection. A primary use for such methods involves the growing demand for automated nucleic acid sequencing. Originally, nucleic acid sequencing involved digestion of the nucleic acids by specific nucleases (restriction enzymes) which cut the polymer adjacent to specific base sequences. The resulting fragments were accurately sized by electrophoresis on agarose gels and then detected through the use of intercalating fluorescent dyes or radioactive probes. The presence of fragments of specific molecular weights could then be used to deduce the sequence of bases in the nucleic acid polymer. To have sufficient material to be readily detected on the gels it was necessary to process relatively large amounts of nucleic acid with relatively large amounts of expensive restriction enzymes. A sensitive automated detection method could greatly speed the process and save money by reducing the need for labor, enzymes and other expensive consumables.
Electrochemical detection is particularly well suited for avoiding the problems of DNA analysis--particularly those caused by sample derivatization and the general problem of limited sample since it uses underivatized samples and can be miniaturized with ease--even to the point of working in nanoliter or even picoliter volumes--without sacrificing sensitivity. To date, most electrochemical detection protocols for nucleic acids have been based on the electroactivity of the nucleobases or the adsorption of single-stranded DNA (ssDNA) to complementary strands immobilized on a electrode surface (this also requires the use of an electroactive molecule that intercalates or otherwise associates with double-stranded DNA (dsDNA)).
Direct electrochemical detection of adenine and guanine bases is possible at mercury, gold, copper and carbon electrodes, where these bases can be oxidized at extremely positive potentials. Although these methods are quite sensitive for nucleic acid bases, high backgrounds and irreversible adsorption of larger molecules lead to poor sensitivity for this approach for the analysis of nucleosides, nucleotides and DNA. In particular, mercury, gold, and carbon surfaces were completely fouled by the adsorption of oligonucleotides and DNA strands. Several investigators subsequently exploited this adsorptive tendency of nucleic acid bases, oligonucleotides and DNA to obtain very sensitive electrochemical detection schemes. These schemes involved the adsorption of the nucleic acid onto the electrode surface in order to concentrate them, and then employed stripping voltammetric procedures to analyze the adsorbed analyte.
Indirect electrochemical detection utilizes electroactive moieties that can label dsDNA. Intercalators bind internally to the double stranded DNA formed at the surface of the electrode, allowing detection of the increased current at the electrode surface due to these species. Alternatively, electrostatic binding of cationic species can occur after intercalation or external binding of an electroactive molecule to DNA, where it can be monitored by electrochemistry of by electrogenerated chemiluminescence. All these methods, however, work on a batch process level, since they require the adsorption of nucleic acids and/or their components to the electrode surface for relatively long period of time (tens of seconds to 10-15 minutes). Therefore, they are not suitable for rapid flow-through detection schemes, such as those that can be coupled to separation methods like liquid chromatography and capillary electrophoresis.
Surprisingly, there have been no reports of the direct electrochemcial measurement of nucleotides or nucleic acids based on the oxidation of their ribose sugar moiety (deoxyribose in the case of DNA). Sugars can be detected at noble metal electrodes by employing pulsed amperometric detection or at electrocatalytic metal (e.g., nickel, lead, gold, copper and similar metals) electrodes by using DC detection. Since detection of sugars is accomplished via an electrocatalytic mechanism, it should also be possible to detect nucleotides via a similar mechanism. In particular, the use of a copper electrode minimizes the possibility of fouling of the electrode surface, since the Cu(II) layer is soluble in high pH buffer, and thus the oxidation of sugars and amines does not cause fouling of the copper surface since the surface is constantly washed off and renewed. Additionally, the potential at the electrode can be continuously cycled as conventionally done in most voltammetric measurements. Unfortunately, voltammetric techniques give poorer detection limits even compared to UV absorbance detection due to the high background charging currents observed when scanning the electrode surface rendering conventional voltammetric methods not very useful for nucleotide or nucleic acid analysis.
The parent to the instant application disclosed new scanning electrochemical methods which effectively decouple the background charging current from the Faradaic current in the frequency domain. This is accomplished by capitalizing on the inherent difference between charging and Faradaic currents. The background or charging currents are mostly linear, and therefore are present primarily at the fundamental excitation frequency. The Faradaic currents are essentially nonlinear at fast scan rates and thus have significant components even in the higher harmonics. By utilizing a sinusoidal excitation waveform, the charging current can be effectively isolated from the Faradaic current signal at higher harmonics, therefore sinusoidal voltammetry can be more sensitive than most traditional electrochemical methods.
The present invention employs a detection approach based on the electrocatalytic oxidation of the sugar backbone present on nucleotides and nucleic acids such as DNA. Electrocatalytic metal surfaces, especially copper surfaces, have been found to catalyze the oxidation of ribose (deoxyribose) sugars, without being fouled by the adsorption of the large DNA strands. This enables the detection of native, underivatized nucleotides, oligonucleotides and DNA strands. Adenine and cytosine, representing the two classes of nucleic acid bases, can be detected with nanomolar detection limits at a copper electrode under the preferred experimental conditions, where the sensitivity for adenine is somewhat higher than that for cytosine. Detection limits for purine-containing nucleotides (e.g., adenosine 5'-monophosphate (AMP), adenosine 5'-diphosphate (ADP), and adenosine 5'-triphosphate (ATP)) are on the order of 70-200 nM. These detection limits are achieved for native nucleotides and are over two orders of magnitude lower than those found with UV absorbance detection. Pyrimidine-based nucleotides could also be detected with high sensitivity due to the presence of the sugar backbone which is electroactive at the copper surface. Because this type of detector is not fouled by the nucleotides, it can be used for sensitive detection of analytes eluting continuously from either a chromatography column or an electrophoresis capillary.
In addition to nucleotides, entire nucleic acid molecules are readily detected. Both single stranded and double stranded DNA were detected with a detection limit in the picomolar concentration range (i.e., 10.sup.-12 moles/L). As the number of ribose sugar moieties increases with the chain length of a nucleic acid polymer, the sensitivity for detection also increases. This facilitates the detection of large DNA strands. Also, all previous detection strategies which are based on electroactivity of the bases face a severe decrease in signal for dsDNA as compared to ssDNA since the bases are on the inside of a double helix where their detection is sterically hindered by the surrounding sugars. In the present invention, the signal from a dsDNA is roughly twice that arising from ssDNA strand of the same length.
Sugars are oxidized at copper (at potentials&gt;+0.4 Volts) due to the electrocatalytic mechanism involving the redox couple Cu(III).fwdarw.Cu(II). Since nucleotides and DNA are large molecules which tend to irreversibly adsorb onto most electrodes, the potential applied to the electrode surface must be scanned through the region at which copper is oxidized. Unfortunately, most electrochemical methods which scan the applied potential do not have sensitivity comparable to DC detection schemes. However, sinusoidal voltammetry (previously disclosed as large amplitude AC voltammetry) is able to detect sugars at a copper surface with very high sensitivity and selectivity compared to existing electrochemical methods.
The electrochemical response can also be characterized in terms of the length of the oligonucleotide and the DNA strands. Frequency domain detection technique is used to detect oligonucleotides, and DNA under experimental conditions similar to those needed for the detection of simple sugars; however, a lower excitation frequency of 2 Hz is preferably used to account for the relatively slower kinetics (i.e., larger molecules) of nucleotides and nucleic acids as compared to those for much smaller mono- and disaccharides. Since nucleotides also contain amine moieties in the nucleobases, and these are also electroactive at a copper surface, some signal from the nucleotides can be contributed by these bases apart from that due to the sugar backbone. Thus, the nature of the nucleobase does change the observed signal both in magnitude and phase angle, and the frequency pattern can be used to differentiate different bases. Thus providing enhanced usefulness of the present invention in automated nucleic acid sequencers by allowing discrimination of base types .